Transiently immortalized cells for use in gene therapy

ABSTRACT

The invention provides methods and compositions for expanding cells that are not abundant or are difficult to obtain in pure form in culture, are in short supply (e.g., human cells), or have brief lifetimes in culture, using fusion polypeptide. The fusion polypeptide has a first region containing a translocation carrier moiety having the function of a transport polypeptide amino acid sequence from, e.g., herpesviral VP22, HIV TAT, Antp HD, Arg repeats, or a cationic polymer, or from homologues or fragments thereof, and a second region with a polypeptide having cell immortalization activity, a polypeptide having telomerase-specific activity, or a polypeptide having telomerase gene activation activity. The resulting cells of the invention are suitable for use in cell therapy.

CLAIM OF PRIORITY

This continuation-in-part application claims priority to United Statesprovisional patent application 60/128,893, filed Apr. 12, 1999; U.S.patent application Ser. No. 09/546,483, filed Apr. 10, 2000, and PCTPublication WO 00/61617, filed Apr. 12, 2000.

FIELD OF THE INVENTION

The invention relates generally to tissue transplantation. Morespecifically, the invention relates to methods of increasing thereplicative capacity of normally quiescent cells, such as normal somaticcells, by transient immortalization or transient telomerization, toproduce cells suitable for cell therapy.

BACKGROUND OF THE INVENTION

Cell therapy is an emerging field for the treatment of medicaldisorders. Cells from various tissue sources have been contemplated fortransplantation into mammals, including human, recipients for treatmentof disease or tissue or organ replacement. Use of primary cells fortransplantation requires continuous access to fresh sources of tissue.This is problematic, however, particularly if human cells are desired.Normal human somatic cells display a finite replicative capacity of50-100 population doublings characterized by a cessation ofproliferation in spite of the presence of adequate growth factors. Thereplicative capacity can be considerably less (10-50 populationdoublings) when these cells are placed in culture in vitro. Thiscessation of replication in vitro is variously referred to as cellularsenescence or cellular aging.

To generate tissue in sufficient quantity for therapeutic use, cells arecommonly immortalized, thus acquiring unlimited replicative capacity andavoiding cellular senescence. The identification of immortalizing genesand development of gene transfer methodologies permit generation of celllines from cell types that are difficult to obtain in sufficientquantity or that have short lifetimes in culture. These immortalizinggenes are typically generated by transfer of a virus or plasmid thatcontain an immortalizing gene. Cell immortalization increases the lifespan of a cell (especially in culture under replicative growthconditions), so that the resulting cell line is capable of beingpassaged many more times than the original primary cells.

The use of immortalized cells in cell therapy, however, can pose seriousrisks for patients, because immortalized cells are in many instancestumorigenic. Moreover, the exogenous DNA containing the nucleic acidcapable of transforming the cells is commonly inserted into cells usinginfectious vectors, such as retroviral vectors. Virally infected cellsalso pose serious risks for patients, such as the potential ofgenerating replication-competent virus during vector production; thepotential recombination between the therapeutic virus and endogenousretroviral genomes, potentially generating infectious agents with novelcell specificities, host ranges, or increased virulence andcytotoxicity; and the potential independent integration into largenumber of cells, increasing the risk of tumorigenic insertional events.

Approaches to avoid these risks have focused on the removal of thegenetic element when differentiation of the target cells is desired. Onesuch approach involves the use of the Cre/loxP recombination system ofbacteriophage P1. In the Cre/loxP procedure, the immortalizing gene, oroncogene, is flanked with recombinase recognition (loxP) sites forinsertion, and subsequently removed via Cre-mediated deletion of theflanked gene segment. However, it is very difficult to prove the absenceof residual immortalizing gene. Any leftover immortalizing gene wouldpose a serious risk to the recipient of the cell therapy, as it mayallow these cells to continue to proliferate in the host aftertransplantation, and form a tumor. Accordingly, tissue from cellsgenerated in this fashion is less desirable for cell therapy.

Thus, a need remains in the art for a method of cell proliferation thatavoids the risks associated with the incorporation of exogenousimmortalization genes in cells to be used for cell therapy.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for generating cellsthat are not abundant, that are difficult to obtain in pure form inprimary culture, that are in short supply (e.g., human cells), or thathave brief lifetimes in culture. The invention relates to methods ofconditional, transient cell proliferation in which immortalization ortelomerization are initiated by the action of exogenously suppliedmolecules and terminated by the removal of these exogenously suppliedmolecules. Cells are proliferated in vitro for cell banking and, uponremoval of the exogenous immortalizing or telomerizing fusion proteins,return to their non-proliferative state. The cells produced by themethods of the invention are suitable for transplantation and celltherapy. The methods of the invention can be used to proliferate anynormally quiescent cell that can be induced to proliferate, such asnormal somatic cells.

Provided in the invention are fusion proteins having a macromoleculartranslocation carrier moiety (“translocation moiety”) coupled to aminoacid sequences from cell immortalization proteins. Examples oftranslocation moieties include, but are not limited to, a transportpolypeptide sequence from herpesviral VP22, a transport polypeptidesequence from human immunodeficiency virus (HIV) TAT, a homeodomain fromthe Antennapedia polypeptide (“Antp HD”), a polymer of L-arginine orD-arginine amino acid residues (“Arg repeats”), a polymer of cationicmacromolecules (“cationic polymer”); or homologues or fragments thereof.Examples of proteins or polypeptides for cell immortalization include,but are not limited to, the 12S and 13S products of the adenovirus E1Agenes, SV40 small T antigen and SN40 large T antigen (and subfragmentsand truncated versions thereof), papilloma viruses E6 and E7, theEpstein-Barr Virus (EBV), Epstein-Barr nuclear antigen-2 (EBNA2), humanT-cell leukemia virus-1 (HTLV-1), HTLV-1 tax, Herpesvirus Saimiri (HVS),mutant p53, and the proteins from oncogenes such as myc, c-jun, c-ras,c-Ha-ras, h-ras, v-src, c-fgr, myb, c-myc, n-myc, and Mdm2. Alsoprovided in the invention are fusion proteins having at least onetranslocation moiety from, e.g., a transport polypeptide amino acidsequence from herpesviral VP22, HIV TAT, Antp HD, Arg repeats, or acationic polymer, or from homologues or fragments thereof, coupled withat least one amino acid sequence from proteins, or fragments thereof,with telomerase-specific activity. Examples of proteins withtelomerase-specific activity include, e.g., telomerase, telomerasereverse transcriptase (TERT), p140, p150, p48, and p43, or homologues orfragments thereof.

In one embodiment of the invention, normally quiescent cells aretransiently immortalized in order to proliferate these cells. Cells ofinterest are cultured in the presence of at least one fusion proteincomprising a first translocation moiety having the transport functionof, e.g., herpesviral VP22 protein, HIV TAT, Antp HD, Arg repeats, or acationic polymer, and a second polypeptide having cell immortalizationactivity. The fusion protein is transported to the nucleus of the cells,and immortalizes the cells. The immortalized cells are expanded in thepresence of at least one fusion protein. Once sufficient cells have beenobtained, the fusion protein is removed from the growth medium, and thecells are cultured until they return to their original differentiated,non-immortalized state.

In another embodiment of the invention, normally quiescent cells areproliferated by transiently telomerizing these cells. Cells of interestare cultured in the presence of at least one fusion protein thatincludes a first component comprising a translocation moiety having thetransport function of, e.g., herpesviral VP22 protein, HIV TAT protein,Antp HD, Arg repeats, or cationic polymer, or fragments or homologsthereof; and a second polypeptide having telomerase-specific activity.The fusion protein is transported to the nucleus of the cells where itsynthesizes telomeric DNA at chromosomal ends, thereby preventingreplicative senescence. The telomerized cells are expanded in thepresence of at least one fusion protein of the invention. Oncesufficient cells have been obtained, the fusion protein is removed fromthe growth medium, and the cells are cultured in a standard fashion inthe absence of the exogenous immortalizing or telomerizing fusionproteins.

In yet another embodiment of the invention, a translocation moiety (suchas VP22, TAT, Antp HD, Arg repeats, or cationic polymer, or fragments orhomologs thereof) and an immortalizing gene (such as hTERT, SV40, etc.)are mixed together and applied to the cells.

In still another embodiment of the invention, normally quiescent cellsare proliferated by transiently telomerizing these cells. Cells ofinterest are cultured in the presence of at least two fusion proteins:(1) a fusion protein comprising a translocation moiety (such as VP22,TAT, Antp HD, Arg repeats, or cationic polymer, or fragments or homologsthereof), and a polypeptide having cell immortalization activity; (2) afusion protein comprising a translocation moiety (such as VP22, TAT,Antp HD, Arg repeats, or cationic polymer, or fragments or homologsthereof), and a polypeptide having telomerase-specific activity. Thecells may be cultured in the presence of more than one type oftranslocation moiety-immortalization fusion or in the presence of morethan one type of translocation moiety-telomerizing fusion, or both. Thecells are expanded in the presence of the fusion proteins untilsufficient cells with increased life span have been obtained. The fusionproteins are subsequently removed from the growth medium, and the cellsare cultured until they return to their original differentiated,non-immortalized state.

The cells produced by the methods of the invention are not permanentlyimmortalized (and thus are not tumorigenic or transformed) and are notvirally infected. Accordingly, these cells, upon removal of theexogenous immortalizing or telomerizing fusion proteins, are suitablefor transplantation and use in cell therapy.

In one embodiment of the invention, cellular immortalization isaccomplished by direct transcriptional activation of telomerase reversetranscriptase, mediated by the addition of VP22-myc fusion protein.Wang, et al., 12 Genes & Dev. 1769-1774 (1998).

In another embodiment of the invention, a specific gene in a cell istransiently activated to specifically activate the expression of theendogenous gene of interest, and express the corresponding protein ofinterest. The proteins of interest include, but are not limited to,human growth hormone (“hGH”), erythropoietin (“EPO”), insulinotropin,insulin, leptin, hGCSF, Factor VIII, Factor VII, Factor IX, Factor X,and tissue-type plasminogen activator (“tPA”).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid map of pVP22-hTERT-1091.

FIG. 2 is a depiction of a set of illustrations showing the detection ofVP22-hTERT and VP22-cMyc chimera proteins by immunocytochemistry (ICC).

FIG. 3 is a Western blot analysis of VP22-based chimera proteinsexpressed in COS, wherein Panel A depicts the VP22-hTERT-(cMyc-HIS-TAG)fusion protein and Panel B depicts the VP22-cMyc-(HIS-TAG) fusionprotein.

FIG. 4A is a graphic depiction of the population doubling curve for1091-MDX01 cell lines, and FIG. 4B is a graphic depiction of. thepopulation doubling curve for mMLV-hTERT immortalized MDX12 (“x” curve)cells compared to the parent primary MDX1 cells (diamond curve) asdetermined by repetitive serial passaging of the VP22-hTERT cell line.

FIGS. 5A and 5B are a set of illustrations of a set of gels showing thetelomerase catalytic activities of VP22-hTERT chimera proteins asdemonstrated by TRAP, in COS cells transiently transfected with VP22,VP22-hTERT, and VP22-hTERT-(cMyc-HIS-TAG) fusion constructs (FIG. 5A) orin stable polyclonal p1091/MDX1 cells, with MDX1 as a negative control,and MDX12 as a positive control (FIG. 5B).

FIGS. 6A-6F are a set of illustrations showing the detection of thepresence of telomerase in p1091/MDX1 by immunocytochemistry (“ICC”).

FIG. 7 is a set of illustrations showing endogenous hTERT and VP22-hTERTmRNA expression as measured by RT-PCR in MDX12 and 1091-MDX01immortalized cell lines.

FIG. 8 is a set of illustrations showing endogenous hTERT and VP22-hTERTmRNA expression as measured by RT-PCR in MDX12 and 1091-MDX01immortalized cell lines.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions for use in conditionalimmortalization of primary mammalian cells. In one embodiment, theinvention reports the coupling of a translocation moiety (including, butnot limited to, VP22, TAT, Antp HD, Arg repeats, or cationic polymer, orfragments or homologs thereof) to an oncogene or immortalizing gene,such that eukaryotic or prokaryotic cells can efficiently produce thefusion protein.

The human telomerase gene (hTERT) is capable of immortalizing primaryhuman fibroblast cells without causing the cells to become“transformed”, (Morales, et al. 1999 Nature Genetics 21: 115-118; Jiang,et al. 1999 Nature Genetics 21: 111-114). In another embodiment, a humantelomerase (hTERT) protein fused to a translocation moiety of theinvention serves a similar immortalizing purpose when the fusionconstruct is carried across the cell membrane of appropriately exposedcells. By analogy, attaching any immortalizing protein, including butnot limited to SV40, cMyc, E1A, E6, E7, ras, etc., to the carboxyterminus of the VP22 protein, or to any other translocation moiety ofthe invention, will allow these resulting fusion proteins to beefficiently transported into the nuclei of exposed primary cells.

The fusion proteins of the invention described herein, including allfusion constructs derived from a translocation moiety of the inventioncoupled to a protein of interest to be transported, such as animmortalizing protein, a telomerizing protein, or a transcription factoror the like that will activate an endogenous gene for an immortalizingprotein or a telomerizing protein, are referred to in combinationhenceforth as “translocation fusion proteins.”

In one embodiment, the invention provides for the conditionalimmortalization of human beta cells (for example, with SV40 TAG, myc,ras, etc.) in a transient fashion (for short periods of time or onlyduring proliferation and expansion of the cells), for as long as thetranslocation moiety-immortalizing protein fusion construct is providedto the cells. Thus, the invention makes conditional immortalizationpossible without the direct use of DNA transfection or viraltransduction.

Supernatants, extracts, or co-cultures from or with the cells producinga fusion construct of the invention can be used to deliver the oncogene,immortalizing protein, telomerase protein, or other protein of interestto the cell of choice. Alternatively, a translocation fusion protein ofthe invention can be purified and added to a culture medium as a mediumsupplement. Cells of interest are then treated with the supplementedculture medium containing the fusion protein additives. To obtain adifferentiated final cell, the fusion protein additives are simplyremoved from the medium, such that culture medium without the fusionprotein additive is provided to the cells.

The advantages of this approach is many. The advantages include theelimination of viral transduction or plasmid transfection of the humancell lines of interest. One can simply add the VP22-fusion proteins as asupplement to the tissue culture growth medium. In this way, the cellswill be directly exposed to the immortalizing signals in culture withoutthe need for permanent genetic modification of the cell with theoncogene or immortalizing gene sequences.

Another advantage of the invention compared to classical immortalizingtechniques is the elimination of gene switch—and Cre/loxinactivation-based systems for the deactivation and elimination,respectively, of the introduced immortalizing gene sequences from thegenome of the thus immortalized mammalian cells. Because noimmortalizing gene nucleic acid sequence is ever introduced into theprimary human cells, there is no need to add complicated gene switches(tet, ecdysone etc.) to turn off the expression of theimmortalizing/transforming oncogene, or to flank the immortalizing genewith loxP sites for later excision of the oncogene by the addition ofCRE recombinase. With the translocation moiety fusion protein systems ofthe invention, one simply removes the proteins responsible for cellexpansion from the tissue culture medium and the cellular transport ofimmortalizing signals is halted. From a safety point of view there isconsiderably less concern about transferring potentially transformedhuman cells into the patient population. By contrast, it is verydifficult with classical gene transfection-transduction methods to provethat the transforming gene sequence has been “completely” eliminated orturned off. In other words, with the traditional gene transformationapproaches currently employed one can never be sure to completelyinactivate every last one of the immortalizing genes originally insertedinto the transplanted cells.

Another advantage of the present invention is that the removal of thetranslocation fusion protein(s) from the expanding cell culture mediumincreases the likelihood of a more efficient and rapid reversion of theexpanding cells to a fully differentiated phenotype.

An additional advantage of the invention is that almost any protein ofinterest can be transported directly to the nucleus, without the needfor gene delivery to the cell. The invention is especially advantageousfor the conditional immortalization of human primary cells, such aspancreatic beta cells, hepatocytes, bone cells cartilage cells,fibroblasts, muscle cells, brain cells or fat cells, or any human cellwhich will benefit from the application of gene therapy techniques. As aresult of efficient nuclear transport (as provided by fusion with atranslocation moiety) the invention can also be used for the transientgene activation of specific endogenous genes in any cell line. When thefusion protein contains polypeptide sequences that transcriptionallyactivate specific gene expression of endogenous proteins, then theinvention can also be used for the production of any protein of interestwith out use of the coding sequence for that gene or protein. Examplesof genes that might be endogenously activated through the use of VP22 orTAT fusion proteins are EPO, Factor VIII, leptin, hGH, insulin, etc.Thus, the invention can be used to transiently gene activate any primaryor immortalized cell, for the production of said protein for any use.

The use of the invention can be transient, can be accomplished with anycell or cell type, and does not require DNA insertion into the promoterregion upstream of the gene. We do not need any sequence informationbeyond that of the specific gene activation proteins. This avoids theuse of gene activation of specific promoter sequences. Moreover, theinvention can be utilized to develop a screen for transcriptionalactivators, which can be fused with a translocation moiety of theinvention to screen for activators of any protein of interest.

In another embodiment, the translocation moiety of the invention iscoupled to telomerase. The VP22-telomerase, TAT-telomerase, AntpHD-telomerase, Arg repeat-telomerase, or cationic polymer-telomerasefusion protein is transiently delivered to the cells of interest. Uponremoval of the translocation fusion protein from the medium, thedelivery process is stopped—but only after transiently “telomerizing”the chromosome tips, and extending replicative cell life for more than50 population doublings or more. This delivery process does not requireretention of the delivered gene sequence in any of our final products.Thus, the invention avoids the retention of viruses, Cre/lox, SV40, ortelomerase in the final cell or device product.

In another embodiment, a translocation moiety of the invention iscoupled to c-myc, which specifically induces telomerase activity viatranscriptional activation, so that there is no need for the telomerasefusion protein (Wang, J. et al. 1998 Genes & Devel. 12, pp. 1769-1774).

Polypeptides having transportfunction. VP22 is a structural proteinfound in Herpes simplex type 1 virus (HSV). The herpesviral HSV-1 virionprotein VP22 possesses an unusual intercellular trafficking mechanism(see PCT International patent application WO 97/05265; Elliott & O'Hare,88 Cell 223-233 (1997)). The protein can efficiently transport itselfthrough the membrane of cells via a non-classical Golgi-independentmechanism. The VP22 protein can transport itself into surrounding cellsas the result of endogenous synthesis and secretion from producing cellsor after exogenous application to naive cells. VP22 can spreadthroughout a monolayer of non-expressing cells, by transportation ofVP22 from the cytoplasm of an expressing cell into neighboring cells.Interestingly, the VP22 protein is naturally targeted to the nucleuswhere it binds directly to chromatin and segregates to daughter cellsafter cell division. Furthermore, when fused to a variety of otherproteins the VP22 protein can transport the fused proteins across cellmembranes, thus carrying the attached proteins into the nucleus. Moreimportantly, the VP22-fused proteins have been shown to retainbiological activity in their chimeric state (i.e., fusion state) and todeliver the activity of the fused polypeptide directly into the exposedcell in a highly efficient manner. This VP22-fusion protein transportcapability has recently been demonstrated for a variety of differentproteins including Green Fluorescent protein, a 27 kDa fluorescentmarker protein, (Elliott & O'Hare, 6 Gene Therapy 149-151, 1999); P-53,a 53 kDa cell cycle regulatory protein, (Phelean et al., 16 NatureBiotechnology 440-443, 1998); Thymidine Kinase, the 52 kDa enzymeserving as the converting enzyme in the pro-drug suicide proteincombination routinely used in gene therapy trials; (Dilber et al., 6Gene Therapy 12-21, 1999), and β-galactosidase, the 116 kDa bacterialenzyme widely employed as a reporter protein in gene expression studies(Invitrogen). In all of these studies the chimeric VP22 10 fusionproteins were efficiently transported into fusion-protein exposed cells.Most importantly, these translocation fusion proteins demonstrated thebiological effects associated with each component of the coupled proteinboth in vitro and also in vivo for the VP220TK system.

Various other proteins have the capability to permeate cellularmembranes by the addition of a membrane-translocating sequence (MTS).See, e.g., Rojas et al., 16 Nature Biotechnology 370-375, 1998. The MTSis a signal peptide with a hydrophobic region (h-region) used to delivervarious peptides and proteins (cargo) across cell membranes in anondestructive manner. See, e.g., Lin et al., 1995 J. Biol. Chem. 270:14255-14258. The MTS construct can additionally include a nuclearlocalization sequence. See, e.g., Lin et al., 1995. HIV-1 TAT (Ensoli etal., 67 J. Virol 277-287 (1993); Fawell et al., 91 Proc. Natl. Acad.Sci. USA 664-668, 1994; Schwarze et al., 285 Science 1569-1572, 1999)and a small number of other non-viral proteins (Jackson et al, 89 Proc.Natl. Acad. Sci. USA 10691-10695, 1992) have also been attributed withintercellular trafficking properties, but none appears to demonstratethis phenomenon as strikingly as VP22, with the exception ofdenatured/renatured TAT protein (U.S. Pat. Nos. 5,652,122; 5,670,617;5,674,980; 5,747,641; and 5,804,604, each of which is incorporatedherein by reference), Antp HD (PCT Publications WO97/12912 andWO99/11809, each incorporated herein by reference), Arg repeats(Canadian Patent No. 2,094,658; U.S. Pat. No. 4,701,521, PCT PublicationWO98/52614, each incorporated herein by reference), and cationicpolymers (U.S. Pat. Nos. 4,701,521 and 4,847,240, each incorporatedherein by reference). A further important property of VP22 is that, whenapplied exogenously to the medium of a cell monolayer, it can be takenup by those untransfected cells, where it accumulates in the cellnucleus.

The term “VP22” refers to protein VP22 of Herpes Simplex Virus (e.g.,HSV1), and transport-active fragments and homologues thereof, includingtransport-active homologues from other herpes viruses includingvaricella zoster virus (“VZV”), equine herpes virus (“EHV”) and bovineherpes virus (“BHV”); modified and mutant proteins and fusionpolypeptides and coupling products having homology therewith and atransport function corresponding to a transport function of VP22 fromHSV1. In context “VP22” also relates to nucleic acid sequences encodingany of the above VP22 polypeptides whether in the form of naked DNA orRNA, or of a vector, or of larger nucleic acid sequences including suchsequences as sub-sequences.

Sub-sequences of herpesviral VP22 protein with transport activity, andmethods of testing these, have been described elsewhere. For example,see PCT International patent applications WO 97/05265, WO 98/04708, andWO 98/32866, each of which is incorporated herein by reference.Sub-sequences of herpesviral VP22 protein with transport activityinclude polypeptides corresponding to amino acids 60-301 and 159-301 ofthe full HSV1 VP22 sequence (aa 1-301). A polypeptide consisting ofamino acid residues (“aa”) 175-301 of the VP22 sequence has markedlyless transport activity, and is less preferred in connection with theinvention. Accordingly, the invention relates in one aspect to coupledand fusion proteins comprising a sub-sequence of VP22 containing asequence starting preferably from about aa 159 (or earlier, towards theN-terminal, in the native VP22 sequence), to about aa 301, and having(relative to the full VP22 sequence) at least one deletion of at leastpart of the VP22 sequence which can extend, for example, from theN-terminal to the cited starting point, e.g., a deletion of all or partof the sequence of about aa 1-158. Less preferably, such a deletion canextend further in the C-terminal direction, e.g., to about aa 175. Forexample, partial sequences in the range from about aa 60-301 to about aa159-301 are preferred.

VP22 sequences, as contemplated herein, extend to homologous proteinsand fragments based on sequences of VP22 protein homologues from otherherpesviruses. For example, corresponding derivatives and VP22-homologuesequences have been obtained from VZV (e.g., all or homologous parts ofthe sequence from aa 1-302), from MDV (e.g., all or homologous parts ofthe sequence from aa 1-249), and from BHV (e.g., all or homologous partsof the sequence from aa 1-258) (see PCT International patentapplications WO 97/05265, WO 98/04708, and WO 98/32866). The sequencesof the corresponding proteins from HSV2, VZV, BHV and MDV are availablein public protein/nucleic acid sequence databases. Thus, for example,within the EMBL/GenBank database, a VP22 sequence from HSV2 is availableas gene item UL49 under accession no. Z86099 containing the completegenome of HSV2 strain HG52; the complete genome of VZV including thehomologous gene/protein is available under accession numbers X04370,M14891, M16612; the corresponding protein sequence from BHV is availableas “bovine herpesvirus 1 virion tegument protein” under accession numberU21137; and the corresponding sequence from MDV is available as geneitem UL49 under accession number LI 0283 for “gallid herpesvirus type 1homologous sequence genes”. In these proteins, especially those fromHSV2 and VZV, corresponding deletions can be made, e.g. of sequenceshomologous to aa 1-159 of VP22 from HSV1. These above cited sequencesare hereby incorporated herein by reference. Homologies between thevarious VP22 polypeptides are readily accessible by the use of standardalgorithms and software, for example those mentioned in PCTInternational patent application WO 95/12673, pg. 9.

Furthermore, chimeric VP22 proteins and protein sequences are alsouseful within the context of the invention, e.g., a protein sequencefrom VP22 of HSV1 for part of which a homologous sequence from thecorresponding VP22 homologue of another herpesvirus has beensubstituted. For example, into the sequence of polypeptide 159-301 fromVP22 of HSV1, C-terminal sequences can be substituted from VP22 of HSV2or from the VP22 homologue of BHV.

Deletion of the 34-amino acid C-terminal sequence from VP22 of HSV1 hasbeen reported to abolish transport-activity (see PCT Internationalpatent applications WO 97/05265, WO 98/04708, and WO 98/32866). Thusthis sequence region contains essential elements for transport activity.According to a preferred embodiment of the invention, there are providedcoupled and fusion polypeptides comprising the 34-amino acid C-terminalsequence from VP22, or a variant thereof, together with a sequence from:(a) a protein or polypeptide for cell immortalization; (b) a protein orpolypeptide that has telomerase-specific activity; or (c) a protein orpolypeptide for the activation of a specific endogenous gene. These areprovided for example for use by administration in the form of protein tocells that will take them up. Coupled products of modified terminalfragments having at least one mutation insertion or deletion relative tothe C-terminal 34 amino acid sequence of HSV1 VP22 are also provided.According to an alternative preferred embodiment of the invention, thefusion polypeptides comprises the 34-amino acid C-terminal sequence fromVP22, or a variant thereof, together with a nucleic acid sequence (DNAor RNA) encoding: (a) a protein or polypeptide for cell immortalization;(b) a protein or polypeptide that has telomerase-specific activity; or(c) a protein or polypeptide for the activation of a specific gene.

Sequences necessary for transport activity have also been reported tocontain one or a plurality of amino acid sequence motifs or theirhomologues from the C-terminal sequence of VP22 of HSV1 or otherherpesviruses, which can be selected from RSASR (SEQ ID NO:3), RTASR(SEQ ID NO:4), RSRAR (SEQ ID NO:5), RTRAR (SEQ ID NO:6), ATATR (SEQ IDNO:7), and wherein the third or fourth residue A can be duplicated,e.g., as in RSAASR (SEQ ID NO:8).

Translocation moieties, as contemplated herein, extend to VP22 sequencesdescribed above and sequences from proteins with translocationproperties similar to VP22. Proteins with translocation propertiessimilar to VP22 include, but are not limited to, the MTS (membranetranslocating sequence) 12 amino acid region from Kaposi fibroblastgrowth factor and a similar 11 amino acid region of the HIV TAT protein(Rojas et al., 16 Nat. Biotechnol. 370-375 (1998) and Schwarze et al.,285 Science 1569-1572 (1999), respectively). Also included are thehomeodomain sequence from Antennapedia (Antp HD, described, e.g., in PCTPublications WO97/12912 and WO99/11809) and sequences containing Argrepeats (described, e.g., in Canadian Patent No. 2,094,658; U.S. Pat.No. 4,701,521; PCT Publication WO98/52614). In addition, cationicpolymers with translocation properties similar to VP22, i.e.,macromolecules that at a neutral pH contain positively charged chemicalgroups sufficient to enhance cellular uptake of molecules covalentlybound to it, are included in the invention. Examples of contemplatedcationic polymers include, but are not limited to, such asmacromolecules that contain sequential portions or spatial arrangementsof positive charges, e.g., homopolymers or copolymers of poly(aminoacids) such as poly-L-lysine, poly-D-lysine, poly-L-arginine,poly-D-arginine, and of non-amino acids such as polysaccharides oruncharged polymers substituted with side chain amines, wherein theresulting positively charge is sufficient to confer on the resultingmacromolecule a translocation property similar to VP22 (see, e.g., U.S.Pat. Nos. 4,701,521 and 4,847,240).

Fusion Proteins. In use, many of the compositions described herein canbe expressed as fusion proteins in a first part of the target populationof cells, exported therefrom, and harvested. The harvested translocationfusion protein is then placed in a growth medium where it is taken up bya second part of the target population of cells not directly producingthe protein.

A translocation fusion polypeptide as described herein can betransported to a target population of cells, by introducing apolynucleotide or other vector encoding the fusion polypeptide into afirst population of cells (e.g., by transfection or microinjection),expressing the encoding polynucleotide to produce the translocationfusion polypeptide, causing it to be exported from the first populationof cells, harvested, and introduced to a second population of cells viathe growth medium.

Translocation moieties of the invention, or functional sub-sequencesthereof, optionally with an additional polypeptide tail for coupling,can be linked to other proteins or nucleic acid by chemical coupling inany known suitable standard manner. Coupling or fusion of an amino acidsequence with the transport function of a translocation moiety, such asVP22 protein, Antp HD, Arg repeat, cationic polymer, or TAT protein, canprovide a useful cell delivery construct for proteins of the kindsmentioned.

The term “fusion proteins” include terms such as “coupled proteins,”“coupling products,” and “fusion products.” Preferably the coupledproteins are fusion proteins, which can conveniently be expressed fromapproximately in-frame-fused gene-coding sequences in known suitablehost cells. Corresponding polynucleotide sequences can be prepared andmanipulated using elements of known and standard recombinant DNAtechniques and readily available adaptations thereof. However,chemically-coupled products can for certain applications be used ifdesired, and can be prepared from the individual protein componentsaccording to any of a variety of chemical coupling techniques known inthe art.

Fusion proteins can be formed and used in ways analogous to or readilyadaptable from standard recombinant DNA techniques. The polynucleotidecan be comprised in an open reading frame operably linked to a suitablepromoter sequence, and form part of an expression vector, e.g.,comprising the polynucleotide carried in a plasmid. The expressionvector can, for example, be a recombinant virus vector or a non-viraltransfection vector. The vectors can, for example, be analogs orexamples of those vectors mentioned or described in PCT Internationalpatent application WO 97/05265, or of those mentioned or described inPCT International patent applications WO 92/05263, WO 94/21807, or WO96/26267. For nucleotide sequences that are capable of being transcribedand translated to produce a functional polypeptide, degeneracy of thegenetic code results in a number of nucleotide sequences that encode thesame polypeptide. The invention includes all such sequences.

Products described herein can be used according to the invention astransportable proteins capable of being taken up by a target populationof cells, e.g., so that an effector function corresponding to thepolypeptide sequence coupled to the translocation moiety, from among thekinds mentioned above, can take place within the target cells that havetaken up the product. Thus, for example, the target cells may beimmortalized in a case where the polypeptide or nucleotide sequence isfrom an immortalizing agent or from an activator of an immortalizingagent, or obtain an extended replicative life span where the polypeptideor nucleotide sequence is from telomerase, or from a telomeraseactivator. In use, many of the products described herein can beexpressed as fusion proteins in a first part of the target population ofcells, exported therefrom, and harvested. The fusion protein is thenplaced in a growth medium where it is taken up by a second part of thetarget population of cells not directly producing the protein.

A translocation fusion protein, as described herein, can be transportedto a first population of cells by introducing a polynucleotide or othervector encoding the fusion polypeptide into a first part of the targetpopulation of cells (e.g., by transfection or microinjection),expressing the encoding polynucleotide to produce the fusionpolypeptide, thereby to cause it to be exported from said first part ofsaid target population, harvested, and introduced to a second part ofthe target population of cells via the growth medium. Fusion protein(including chemically coupled products) can also be transported into atarget population of cells by directly exposing the cells to apreparation of the fusion protein, thereby to cause the target cells totake them up.

Among the derivatives of VP22 that can be used according to aspects ofthe invention as transport active substances and for coupling withmaterials to be transported, for the purposes set forth elsewhereherein, are peptides comprising a transport-active functional sequencefrom the C-terminal section of VP22. Among the derivatives ofAntennapedia that can be used according to aspects of the invention astransport active substances and for coupling with materials to betransported, for the purposes set forth elsewhere herein, are peptidescomprising a transport-active functional sequence from the homeodomainsection of Antennapedia. Among the derivatives of an Arg repeat that canbe used according to aspects of the invention as transport activesubstances and for coupling with materials to be transported, for thepurposes set forth elsewhere herein, are peptides containing atransport-active functional sequence of contiguous or partiallycontiguous segments of at least 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25,50, 100, or 1000 arginine residues, wherein the arginine residues may beof the D-form, the L-form, or mixtures of each. Likewise, derivatives ofcationic polymers with the above functions may contain transport-activefunctional sequence of contiguous or partially contiguous segments of atleast 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 50, 100, or 1000 lysineresidues, wherein the lysine residues may be of the D-form, the L-form,or mixtures of each. The above described Arg repeats, cationic polymersof lysine residues, or both, may be expressed from a nucleic acidencoding the desired residues coupled with a second nucleic acidsencoding the remaining component to create a fusion protein of theinvention, or they may be synthesized and conjugated to a secondcomponent of the invention to create the fusion protein of interest.

In an example embodiment, the coupling products or fusion proteins basedon VP22 can have a range of molecular sizes. The products can inpractice be for example up to about 70 kDa or more, e.g., 90 kDa or 100kDa or more in respect of the size of the protein to be coupled or fusedto VP22. The embodiments of the invention include examples where thefusion peptide is at least about 13 residues long, or more than about 12amino acid residues long. The proteins to be fused can sometimes also bemore than about 27 or 32 kDa. The coupled polypeptide or fusion protein,including the VP22 component can have sizes greater than 120 kDa, e.g.,up to about 180 kDa or 200 kDa.

It is sometimes preferred that the translocation moiety sequence isfused at its N-terminus to the sequence of the chosen other protein ofone of the kinds mentioned herein. Alternatively, C-terminal fusions ofthe translocation moiety can sometimes be correspondingly preferred.

In the translocation fusion polypeptides of the invention, mutations ofthe constituent amino acid sequences can be incorporated in the fusionpolypeptides and other coupled proteins. Included here are proteinshaving mutated sequences such that they remain homologous, e.g. insequence, function; and antigenic character or other function, with aprotein having the corresponding parent sequence. Such mutations canpreferably for example be mutations involving conservative amino acidchanges, e.g., changes between amino acids of broadly similar molecularproperties. For example, interchanges within the aliphatic groupalanine, valine, leucine and isoleucine can be considered asconservative. Sometimes substitution of glycine for one of these canalso be considered conservative. Interchanges within the aliphatic groupaspartate and glutamate can also be considered as conservative.Interchanges within the amide group asparagine and glutamine can also beconsidered as conservative. Interchanges within the hydroxy group serineand threonine can also be considered as conservative. Interchangeswithin the aromatic group phenylalanine, tyrosine and tryptophan canalso be considered as conservative. Interchanges within the basic grouplysine, arginine and histidine can also be considered conservative.Interchanges within the sulfur-containing group methionine and cysteinecan also be considered conservative. Sometimes substitution within thegroup methionine and leucine can also be considered conservative.Preferred conservative substitution groups are aspartate-glutamate;asparagine-glutamine; valine-leucine-isoleucine; alanine-valine;phenylalanine-tyrosine; and lysine-arginine. In other respects, mutatedsequences can comprise insertions such that the overall amino acidsequence is lengthened while the protein retains transport properties.Additionally, mutated sequences can comprise random or designed internaldeletions that shorten the overall amino acid sequence while the proteinretains transport properties.

The mutated protein sequences can additionally or alternatively beencoded by polynucleotides that hybridize under stringent conditionswith the appropriate strand of the naturally-occurring polynucleotideencoding the parent protein, and can be tested for positive results inknown functional tests relevant to the parent protein. ‘Stringentconditions’ are sequence dependent and will be different in differentcircumstances. Generally, stringent conditions can be selected to beabout 5° C. lower than the thermal melting point (T_(M)) for thespecific sequence at a defined ionic strength and pH. The T_(M) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Typically,stringent conditions will be those in which the salt concentration is atleast about 0.02 molar at pH 7 and the temperature is at least about 60°C. As other factors may affect the stringency of hybridization(including, among others, base composition and size of the complementarystrands), the presence of organic solvents and the extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one.

Translocation fusion proteins of the invention can be easily purifiedusing a poly-His (poly-histidine) tag. Systems for producing fusionproteins containing poly-His tags are commercially available (Xpress,Invitrogen, San Diego Calif.; HisTrap kit, Pharmacia Biotech Inc.,Piscataway, N.J.).

Coupling with immortalizing proteins. A common approach to lengtheningthe life span of a cell is to transfer a virus or a plasmid thatcontains one or more immortalizing genes. Cell immortalization increasesthe life span of a cell, and the resulting cell line is capable of beingpassaged many more times than the original cells. However, irreversiblytransformed, tumorigenic human cells may form colonies in soft agar andalso may form tumors in nude mice. These cells are unsuitable for directcell therapy or any assay of fully differentiated cell functions.

In one useful class of embodiments of the invention, VP22 can be coupledwith known proteins or polypeptides that cause cells to be immortalized.Immortalizing genes are well known in the art. See, e.g., Katakura etal., Methods Cell Biol. 57: 69-91 (1998). Immortalizing proteins orpolypeptides include, but are not limited to, the 12S and 13S productsof the adenovirus E1A genes, SV40 small and large T antigens, papillomaviruses E6 and E7, the Epstein-Barr Virus (EBV), Epstein-Barr nuclearantigen-2 (EBNA2), human T-cell leukemia virus-1 (HTLV-1), HTLV-1 tax,Herpesvirus Saimiri (HVS), mutant p53, and the proteins from oncogenessuch as myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src, c-fgr, myb, c-myc,n-myc, and Mdm2.

In an example of the invention concerned with cell immortalization, VP22is coupled with the oncoprotein myc (v-myc or c-myc). Thus, according toone embodiment of the invention, there is provided a fusion proteincomprising an amino acid sequence with the transport function ofherpesviral VP22 protein and a sequence with the immortalizationfunctionality of myc. In a preferred embodiment, the fusion proteinincludes substantially the full length myc sequence and substantiallythe full length VP22 sequence.

Fusion with a translocation moiety of the invention is useful fordelivery of an agent for immortalization such as myc. Where thedescription given herein refers to myc and related peptides; it will beunderstood that, where the context admits, alternative immortalizingagents, such as for example those myc analogues and other immortalizingagents mentioned and referred to herein, are also contemplated, as are,more generally, alternative fusion or coupling partners fortranslocation moieties, of any of the other types mentioned herein. Onceexpressed in a subpopulation of expressing cells, harvested, andadministered to the growth medium of cells of interest, such a fusionprotein can be transported by the translocation moiety's transportmechanism to a significant portion of the target normal somatic cells soexposed, and the foreign attached polypeptide will then transientlyimmortalize these normal cells.

Also provided by this aspect of the invention are correspondingpolynucleotides encoding a fusion polypeptide that include a sequencewith the transport function of a translocation moiety of the invention,e.g., herpesviral VP22 protein, and a sequence with the human or othermammalian cell proliferating function of an oncogene, e.g., myc. Thepolynucleotide can be created in an open reading frame operably linkedto a suitable promoter sequence.

The resulting polynucleotide forms an “expression cassette” and can,according to contemplated examples of the invention, form part of anexpression vector, e.g., comprising the polynucleotide carried in aplasmid. The expression vector can be for example a virus or a non-viraltransfection vector.

Coupling with telomerase. Telomeres are specialized structures at theends of eukaryotic chromosomes and appear to function in chromosomestabilization, positioning, and replication. Blackburn & Szostak, 53Ann. Rev. Biochem. 163-194 (1984); Zakian, 23 Ann. Rev. Genetics 579-604(1989); Blackburn, 350 Nature 569-573 (1991). In all vertebrates,telomeres consist of hundreds to thousands of tandem repeats of5′-TTAGGG-3′ sequence and associated proteins (Blackburn 1991 Nature350: 569-573; Moyzis et al., 1988 Proc. Natl. Acad. Sci. 85: 6622-6626).Southern blot analysis of chromosome terminal restriction fragments(TRF) provides the composite lengths of all telomeres in a cellpopulation (Harley et al., 3445 Nature 458-460 (1990); Allsopp et al.,89 Proc. Natl. Acad. Sci. USA 10114-10118 (1992); Vaziri et al., 52 Am.J Human Genetics 661-667 (1993)). In all normal somatic cells examinedto date, TRF analysis has shown that the chromosomes lose about 50-200nucleotides of telomeric sequence per cell division, consistent with theinability of DNA polymerase to replicate linear DNA to the ends (Watson,239 Nature New Biology 197-201 (1972)).

This shortening of telomeres has been proposed to be the mitotic clockby which cells count their divisions (Harley, 256 Mut. Res. 271-282(1991)), and a sufficiently short telomeres may be the signal forreplicative senescence in normal cells (Hastie et al., 346 Nature866-868 (1990); Lindsey et al., 256 Mut. Res. 45-48 (1991); Wright &Shay, 8 Trends Genetics 193-197 (1992)). In contrast, the vast majorityof immortal cells examined to date show no net loss of telomere lengthor sequence with cell divisions, suggesting that maintenance oftelomeres is required for cells to escape from replicative senescenceand proliferate indefinitely (Counter et al., 11 EMBO 1921-1929 (1992);Counter et al., 91 Proc. Natl. Acad. Sci. USA 2900-2940, 1994).

Telomerase, a unique ribonucleoprotein DNA polymerase, is the onlyenzyme known to synthesize telomeric DNA at chromosomal ends using as atemplate a sequence contained within the RNA component of the enzyme(Greider & Blackburn, 43 Cell 405-413 (1985); Greider & Blackburn, 337Nature 331-337 (1989); Yu et al., 344 Nature 126-132 (1990); Blackburn,61 Ann. Rev. Biochem. 113-129 (1992)). With regard to human cells andtissues, telomerase activity has been identified in immortal cell linesand in ovarian carcinoma but has not been detected in mortal cellstrains or in normal non-germline tissues (Morin, 59 Cell 521-529,1989). Together with TRF analysis, these results suggest telomeraseactivity is directly involved in telomere maintenance, linking thisenzyme to cell immortality.

Expression of the human telomerase catalytic component (hTERT) hasrecently been studied in human somatic cells. Jiang, et al., 21 NatureGenetics 111-114 (1999). Telomerase expression in normal somatic cellsdid not appear to induce changes associated with a malignant phenotypesuch as abnormal growth control or oncogenic transformation. The absenceof cancer-associated changes was also reported in human fibroblastsimmortalized with telomerase. Morales, et al., 21 Nature Genetics115-118 (1999). It was demonstrated that the introduction of telomeraseinto normal human somatic cells does not lead to growth transformation,does not bypass cell-cycle induced checkpoint controls and does not leadto genomic instability of these cells. Methods for detecting telomeraseactivity, as well as for identifying compounds or polypeptides thatregulate or affect telomerase activity, together with methods fortherapy or diagnosis of cellular senescence and immortalization bycontrolling or measuring telomere length and telomerase activity, havealso been described (see PCT International patent application WO93/23572). The identification of compounds affecting telomerase activityprovides important benefits to efforts at treating human disease.Compounds that stimulate or activate telomerase activity (such as myc)can be fused to translocation moieties of the invention to inhibit cellsenescence and increase the life span of the cell.

In a further class of embodiments of the invention, translocationmoieties of the invention, or functional subsequences thereof, can beusefully coupled or fused with telomerase, or other enzyme or functionalfragment thereof known as applicable for a similar purpose. The couplingproduct can penetrate into cells that are to be treated. Suchtranslocation fusion proteins made up of translocation moiety-telomerasecoupling products are used in the extension of the replicative life spanof the target cell. The telomerase functions to stabilize the telomeresof the cell and arrest cellular senescence. See, e.g., U.S. Pat. No.5,837,857.

Transient immortalization or telomerization of cells. According to oneaspect of the invention, normally quiescent cells are transientlyimmortalized in order to proliferate these cells. Cells of interest arecultured in the presence of a fusion protein comprising a firstpolypeptide having: (1) a translocation moiety and (2) a polypeptidehaving cell immortalization activity. The resulting fusion protein istransported to the nucleus of the cells, and immortalizes the cells. Theimmortalized cells are expanded in the presence of the fusion protein.Once sufficient cells have been obtained, the fusion protein is removedfrom the growth medium, and the cells are cultured until they return totheir original, non-immortalized state. More than one oncogene orimmortalizing fragment may be required.

According to another aspect of the invention, normally quiescent cellsare proliferated by transiently telomerizing these cells. Cells ofinterest are cultured in the presence of a fusion protein comprising:(1) a translocation moiety and (2) a polypeptide havingtelomerase-specific activity. The resulting fusion protein istransported to the nucleus of the cells where it synthesizes telomericDNA at chromosomal ends, thereby preventing replicative senescence. Oncesufficient cells have been obtained, the translocation fusion protein isremoved from the growth medium, and the cells are cultured according tostandard techniques, in absence of the fusion proteins.

According to yet another aspect of the invention, normally quiescentcells are proliferated by transiently immortalizing and telomerizingthese cells. Cells of interest are cultured in the presence of twofusion proteins: (1) a fusion protein comprising a first part containinga translocation moiety of the invention and a second part containing apolypeptide having cell immortalization activity; and (2) a fusionprotein comprising a first part containing a translocation moiety of theinvention and a second part containing a polypeptide havingtelomerase-specific activity, or telomerase gene activation activity.The cells are expanded in the presence of the fusion proteins untilsufficient cells have been obtained. The cells are then subsequentlycultured according to standard techniques, in absence of the fusionproteins.

Transient gene activation. In another embodiment of the invention, aspecific gene in a cell is transiently activated to specificallyactivate the expression of the endogenous gene of interest. The genes ofinterest include, but are not limited to, hGH, FAC VIII, FAC VII, FACIX, FAC X, EPO, insulin, and TPA.

Equivalents. From the foregoing detailed description of the specificembodiments of the invention, it should be apparent that unique methodsof increasing the replicative capacity of normally quiescent cells havebeen described. Although particular embodiments have been disclosedherein in detail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims which follow. In particular, it iscontemplated by the inventor that various substitutions, alterations,and modifications may be made to the invention without departing fromthe spirit and scope of the invention as defined by the claims. Forinstance, the choice of the particular translocation moiety having thetransport function similar to herpesviral VP22 protein, or theparticular polypeptide having cell immortalization activity, or theparticular polypeptide having telomerase-specific activity, is believedto be a matter of routine for a person of ordinary skill in the art withknowledge of the embodiments described herein.

The details of one or more embodiments of the invention have been setforth in the accompanying description above. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural referents unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All patents and publicationscited in this specification are incorporated by reference.

The following EXAMPLES are presented in order to more fully illustratethe preferred embodiments of the invention. These EXAMPLES should in noway be construed as limiting the scope of the invention, as defined bythe appended claims.

EXAMPLE 1 Construction of the VP22-hTERT FUSION

Materials. Taq polymerase and all restriction, modifying, and enzymeswere purchased from Life-Technologies (Basel, Switzerland). Theexpression vector pCEP4, TA- and Cloning kits were obtained fromInvitrogen Corporation (Carlsbad, Calif., U.S.A.). The Quick-Clone CDNAfrom human testis, lymphoma and HeLa cells, GC-Melt Genomic and cDNA PCRkits were purchased from ClonTech (Basel, Switzerland). The TRAPezeassay kit was obtained from Oncor (Basel, Switzerland).

Oligonucleotides. The following oligonucleotides were custom synthesized(Life-Technologies or Microsynth) for use as PCR primers in the cloningof the hTERT cDNA:

5′-ATATATGCTAGCGCCACCATGCCGCGCGCTCCCCGCTGCC-3′ (SEQ ID NO:1).

5′-ATATATGAATTCAGTCCAGGATGGTCTTGAAGTCTGAGGGC-3′ (SEQ ID NO:2).

RT-PCR amplification of 293T CDNA using Taq polymerase with theseprimers produced a 3417 base-pair product. Diagnostic restrictiondigestion patterns confirmed that this 3417-bp RT-PCR product was indeedderived from the hTERT cDNA. The product was subsequently subcloned intopCR2.1 by using the TOPO-TA cloning kit (Invitrogen). Nucleotidesequence determination of the resulting clone, pCR2.1-hTERT-1007,demonstrated that the cloned hTERT had the correct nucleotide sequencewhen compared to the published hTERT nucleotide sequence. Nakamura etal., 1997; GenBank Accession Number AF05950.

Construction of the VP22-hTERT Chimera Fusion Gene. A 3416-bp EcoRIfragment containing the intact hTERT cDNA was excised out ofpCR2.1-hTERT-1007 and subcloned, in-frame, at the EcoRI site in theexpression vector pVP22-1090 (Invitrogen). The resulting clone was namedpVP22-hTERT-1091, as shown in FIG. 1. In FIG. 1, the black arrows withinthe circular map represent the VP22 and hTERT coding regions that willbe expressed as a chimeric fusion protein. The backbone plasmid (pVP22plasmid) was purchased from InVitrogen.

Generation of p1091/BHK Stable Cell Line. The pVP22-hTERT-1091expression vector was stably transfected into BHK cells by usingLipofectamine Plus (Gibco Life-Technology). Briefly, the selection drugG418 was added to the transfected BHK cells 24 hours after transfection.Stably transfected p1091/BHK cells were grown in fibroblast growth media(DMEM plus 10% FBS) and 1 mg/ml of G418 for 2 weeks. One million ofp1091/BHK cells were seeded in one T-75 flask and pulsed two times with12 ml of DMEM plus 10% FBS for 24 hours. The collected, conditionedmedia were filter-sterilized through a Nunc 0.22 μm-filter and overlaidon to primary human fibroblasts, MDX1 (Modex thérapeutiques SA), at50,000 cells per well in a 6-well plate. Final concentrations offibroblast growth media were either 25%, 50%, 75%, or 100%. After thefirst 24 hours of exposure to the p1091/BHK conditioned media, the mediawere removed and replaced with the second batch of a 24-hour pulsedp1091/BHK conditioned media. After the second 24-hour exposure, MDX1cells were harvested by trypsinization and pelleted.

Telomerase Assay. The Telomeric Repeat Amplification Protocol (TRAP)assay was performed according to the manufacturer's protocol (TRAPezeTelomerase Detection Kit, Oncor). Briefly, a pellet of 50,000 cells wasresuspended in 50 μl of 1× CHAP lysis buffer containing RNaseOut at 200U/ml (Gibco Life-Technology). The cell suspension was incubated on icefor 30 minutes and immediately centrifuged at 15,000 RPM at 4° C. for 15minutes. T supernatant was immediately transferred to an RNase-FreeEppendorf tube. According to the manufacturer's protocol, the cellextract was diluted 1:10 so that a cell extract from 200 cells was usedfor the TRAP assay. Ten microliters of the reaction mix were resolvedvia 12.5% non-denaturing PAGE in 0.5× TBE buffer at 150 volts for 2hours. The DNA ladders were visualized by staining in SYBR Green Stain(Molecular Probe).

Results. MDX1 primary human fibroblasts do not possess detectabletelomerase enzyme activity as monitored by the TRAP assay. Thus, anydetected telomerase enzyme activity from the MDX1 cells exposed to theconditioned media from pVP22-hTERT-1091/BHK can be attributed to thechimeric VP22-hTERT proteins that had been secreted by the stablepVP22-hTERT-1091/BHK cells and subsequently taken up by the primaryhuman fibroblast MDX1 cells. PAGE results showed the telomeraseactivities from MDX1 cells that were exposed to the conditioned mediafrom pVP22-hTERT-1091/BHK cells. The positive ladder formation in the50% media mix indicated that the chimeric VP22-hTERT secreted from thepVP22-hTERT-1091/BHK cells was taken up by the primary human fibroblastMDX1.

EXAMPLE 2 Transient Immortalization Technology

The goal of this EXAMPLE is to validate the feasibility of chimericprotein translocation systems for the transient immortalization ofprimary human cells.

Construction of VP22-hTERT Fusion Cassettes and Expression Vectors: Thebasic VP22 expression vector was purchased from Invitrogen and renamedas pVP22-(cMyc-HIS-TAG)-1091. The cMyc and HIS denote the cMyc-tags andHIS-tags that are fused at the C-terminus of the VP22. The first gene tobe fused to the VP22 was chosen to be the hTERT that was usedsuccessfully to enhance the proliferative potential of the primary humanfibroblasts. Due to the concern that the C-terminal tags might interferethe hTERT catalytic activity, it was decided to make two VP22-hTERTfusion cassettes: (1) VP22-hTERT contains an in-frame fusion betweenVP22 and hTERT with no cMyc and HIS tags at the C-terminus of the fusionprotein and (2) VP22-hTERT(cMyc-HIS-TAG) contains an in-frame fusionbetween VP22 and hTERT with cMyc and HIS tags at the C-terminus of thefusion protein. The cMyc and HIS tags were included at the N-terminusdue to the need to identify the fusion protein inside mammalian cellsand to be able to purify the fusion protein in large quantities.

The 3.4-kb EcoRI insert containing the hTERT was excised out of thep1007 and subcloned into the pVP22-cMyc-HIS-1090 at the EcoRI siteresulting in an in-frame fusion between the N-terminal VP22 and theC-terminal hTERT. This new construct was named pVP22-hTERT-1091 (see,TABLE 1).

TABLE 1 A list of VP22-based fusion genes. (cMyc-HIS-TAG) denotes thecMyc-epitope and HIS-epitope tags fused in-frame at the C-terminus ofthe VP22, VP22-hTERT, and VP22-cMyc fusion genes. Expression CassetteSequence Plasmid Description Verified Western ICC TRAP 1091 VP22-hTERT +− + + 1095 VP22-hTERT- + + + + (cMyc-HIS-TAG) 1101 VP22-cMyc-(HIS- + + +− TAG)

To generate the VP22-hTERT(cMyc-HIS-TAG) fusion protein, we removed thetermination codon TGA at the end of the hTERT coding sequence. Briefly,the 3′ 900-bp MluI/NotI fragment in hTERT was regenerated by PCR usingoligonucleotides in order to generate a 3′ 900-bp MluI/NotI fragmentcontaining no TGA termination codon. This 3′ 900-bp MluI/NotI hTERT PCRproduced with no TGA termination codon was cloned directly intopCR2.1-TOPO cloning vector and the resulting plasmid was named aspCR2.1-TOPO-hTERT(−)TGA-1093. The nucleotide validity of the PCR insertin p1093 was confirmed to be 100% accurate by nucleotide sequencingreactions. The 3′ 900-bp MluI/NotI fragment in pVp22-hTERT-1091 wassubstituted with the one from p1093 resulting in an in-frame fusion ofVP22, hTERT, cMyc epitope tag, and HIS epitope tag. The resultingexpression vector was named as pVP22-hTERT-(cMyc-HIS-Tag)-1095.

Construction of VP22-cMyc Fusion Cassettes and Expression Vectors: TheVP22-cMyc fusion cassette contains only HIS-tag at the C-terminus of thefusion protein. In order to easily fuse in-frame the VP22 and cMyccoding sequences, the entire cMyc coding sequence was regenerated in a5′ 790-bp and 3′ 549-bp fragments by resulting in pCR2.1-cMyc-PCR1-1097and -1099, respectively. The nucleotide validity of the PCR inserts inp1097 and p1099 was confirmed to be 100% accurate by nucleotidesequencing reactions. The 5′ 790-bp and 3′ 549-bp cMyc fragments werecombined by subcloning the 3′ 549-bp ClaI/BamHI from p1099 into theClaI/BamHI site in the p1097 generating a full-length cMyc without a TGAtermination codon. This plasmid was named aspCR2.1-TOPO-cMyc-(−)TGA-1100. Subsequently, the full-length cMyc withoutthe TGA termination codon was subcloned out of p 1100 by EcoRI and NotIrestriction digestion and into p1090 generating pVP22-cMyc-HIS-1101.

Detection of VP22-hTERT and VP22-cMyc Fusion Proteins by ICC Analysis:To demonstrate that the VP22-hTERT and Vp22-cMyc fusion genes wereproperly constructed, immunocytochemistry (ICC) analysis was performedon COS cells that were transiently transfected with either p1095 orp1101. The control with no DNA plasmid (see, FIG. 2, Panels A and B) andthe control VP22 vector pVP22-(cMyc-HIS-TAG)-1090 was included (see,FIG. 2, Panels C and D). Since VP22, hTERT and cMyc are nuclearproteins, clear nuclear staining of p1090, p1095- and p1101-transientlytransfected was clearly demonstrated by ICC using anti-cMyc antibodies.This data also showed that the fusion VP22-hTERT (see, FIG. 2, Panels Eand F) and VP22-cMyc (see, FIG. 2, Panels G and H) fusion genes werecorrectly constructed and the respective fusion proteins were properlysynthesized and transported to the nucleus as expected of nuclearproteins. Results in FIG. 2 were obtained by transiently transfectingCos cells with no plasmid DNA (Panels A and B),pVP22-(cMyc-HIS-TAG)-1090 control vector (Panels C and D),pVP22-hTERT-(cMyc-HIS-TAG)-1095 vector (Panels E and F), andpVP22-cMyc-(HIS-TAG)-1101 vector (Panels G and H). Primary anti-cMycantibody (c-Myc Ab) was added in Panels A, C, E, and G whereas noprimary anti-cMyc antibody was added in Panels B, D, F, and H.

Detection of VP22-hTERT and VP22-cMyc Fusion Proteins by Western BlotAnalysis: To demonstrate that the VP22-hTERT and Vp22-cMyc fusionproteins were of the correct molecular weight sizes, we performedWestern blot analysis on total cell lysates from COS cells that weretransiently transfected with either p1095 or p1101. The fusion proteinswith the expected molecular weights were clearly detected by anti-cMycantibodies, thus, further validating that the fusion VP22-hTERT (see,FIG. 3, Panel A) and VP22-cMyc (see, FIG. 3, Panel B) fusion genes wereconstructed correctly. In FIG. 3 Panel A, the VP22-hTERT-(cMyc-HIS-TAG)fusion protein and the control VP22-(cMyc-HIS-TAG) protein in expressionvectors p1095 and p1090, respectively, were expressed in transientlytransfected COS cells. The primary antibody used was the anti-cMycantibody from Invitrogen. In FIG. 3 Panel B, the VP22-cMyc-(HIS-TAG)fusion protein and the control VP22-(cMyc-HIS-TAG) protein in expressionvectors p1101 and p1090, respectively, were expressed in transientlytransfected COS cells. The primary antibody used was the anti-cMycantibody from Invitrogen. The arrows molecular weights ofVP22-hTERT-(cMyc-HIS-TAG), VP22-cMyc-(HIS-TAG), and TAG).

Demonstration of enhanced proliferation capacity: The VP22-hTERT stablytransfected cell line demonstrates a fully immortalized phenotype asevidenced by the enhanced population doubling curve depicted in FIG. 4.FIG. 4A shows the population doubling curve for 1091-MDX01 as determinedby repetitive serial passaging of the VP22-hTERT cell line. FIG. 4Bshows the population doubling curve for mMLV-hTERT immortalized MDX12(“x” curve) compared to the parent primary MDX1 cell (diamond curve) asdetermined by repetitive serial passaging of the VP22-hTERT cell line.Normal non-immortalized primary MDX01 cells demonstrate 13-20 PD's underthe same culture conditions.

Demonstration of the Catalytic Enzyme Activity by VP22-hTERT FusionProteins: To demonstrate that the N-terminal VP22 fusion protein did notaffect the catalytic activity of the C-terminal fused hTERT enzyme, TRAPenzyme assays were performed on total cell extracts from telomerasenegative MDX1 cells that were transiently transfected with either p1091or p1095. Both p1091 and p1095 exhibited clear ladder formation clearlyindicating the preservation of the telomerase catalytic activity in theVP22-hTERT fusion proteins (see, FIG. 4A).

To determine if VP22-hTERT fusion protein encoded by expression plasmidp1091 was capable of immortalizing telomerase negative primary humanfibroblast cell lines these constructs were stably transfected intohTERT-negative MDX1 cells. After 5 weeks of G418 selection at 50 ng/ml,a small colony of G418-resistant p1091/MDX1 was selected and expandedfor further population doubling studies. TRAP assay on p1091/MDX1clearly showed the hTERT enzyme activity (see, FIG. 5B). In FIG. 5A,TRAP assay was performed on cell lysates from COS cells transientlytransfected with VP22, VP22-hTERT, and VP22-hTERT-(cMyc-HIS-TAG) fusionconstructs in expression vectors p1090, p1091, and p1095, respectively.293T was included as positive control for hTERT catalytic activities inTRAP. In FIG. 5B, TRAP assay was performed on stable polyclonalp1091/MDX1 cells, MDX1 as negative control, and MDX12 as positivecontrol.

Furthermore, ICC using anti-hTERT antibodies clearly demonstratedpositive nuclear staining in p1091/MDX1 cells (see, FIG. 6A through FIG.6F). ICC was performed on p1091/MDX1 stable cells with anti-hTERTantibody, -hTERT(C-20) Ab, while MDX1 (FIG. 6A and 6B) and MDX12 (FIG.6C and FIG. 6D) cells were used as hTERT-negative and hTERT-positivecontrols, respectively. Primary anti-hTERT antibody was added in 6A, 6C,and 6E whereas no primary anti-hTERT antibody was added in 6B, 6D, and6F. These results confirm the presence of “telomerase” proteins inp1091/MDX1 and are consistent with the detection of telomeraseactivities in p 1091/MDX1 by TRAP assays. P1091/MDX1 stable cells wereshown to be G418-resistant but Hygromycin-sensitive demonstrating thatthey are not “contaminant” cells derived from MDX12 line which is aHygromycin-resistant mMMLV hTERT immortalized fibroblast positivecontrol cell line.

However, there is still a possibility that the p1091/MDX1 cells arederived from spontaneously transformed MDX 1 cells instead of beingtruly immortalized due to the forced expression of VP22-hTERT fusionproteins. To resolve this issue, RT-PCR analysis was performed on totalmRNA isolated from MDX01, p1091-MDX01 (VP22-hTERT immortalized), andMDX12 (mMLV-hTERT immortalized) cell lines. As shown in the left handside of FIG. 7, RT-PCR was performed on mRNA isolated from MDX12 (hTERTimmortalized) and 1091-MDX01 (VP22-hTERT immortalized)cell lines usingoligonucleotides specific for endogenous hTERT mRNA expression. As shownin the right hand side of FIG. 7, RT-PCR was performed on mRNA isolatedfrom MDX12 (hTERT immortalized) and 1091-MDX01 (VP22-hTERT immortalized)cell lines using oligonucleotides specific for VP22-hTERT mRNAexpression (FIG. 7B). +RT signifies addition of reverse transcriptaseand −RT signifies PCR without reverse transcriptase addition; +RT*indicates use of a different brand of reverse transcriptase. As shown inFIG. 7, when RT-PCR is performed using an oligonucleotide pair specificfor the 5′ untranslated portion and the coding regions of the endogenoushTERT gene, no PCR signal is obtained from the mMLV-hTERT or VP22-hTERTimmortalized cell lines. When RT-PCR is performed with a pair ofoligonucleotides specific for the VP22 and hTERT coding regions only theVP22-hTERT immortalized 1091-MDX01 cell line is positive.

Furthermore, in FIG. 8A, further confirms that when RT-PCR is performedusing oligonucleotide pairs specific to the mMLV and hTERT codingregions only the mMLV-hTERT immortalized MDX12 cell line demonstrates apositive signal. In FIG. 8, RT-PCR was performed on mRNA isolated fromMDX12 (hTERT immortalized) and 1091-MDX01 (VP22-hTERT immortalized) celllines using oligonucleotides specific for mMLV-hTERT mRNA expression(FIG. 8, top). RT-PCR was performed on mRNA isolated from MDX12 (hTERTimmortalized) and 1091-MDX01 (VP22-hTERT immortalized)cell lines usingoligonucleotides specific for VP22-hTERT mRNA expression (FIG. 8,bottom). +RT signifies addition of reverse transcriptase and -RTsignifies PCR without reverse transcriptase addition; +RT* indicates useof a different brand of reverse transcriptase.

Thus, the identity of the telomerase enzyme activity as detected by TRAPon p1091/MDX01 cell extracts can be attributed to the VP22-hTERT fusionmRNA and not to endogenously activated hTERT proteins. This demonstratesthat similar to MDX01 cells immortalized with constitutively expressedmMLV-hTERT the VP22-hTERT immortalized 1091-MDX01 is also fullyimmortalized by the expression of the VP22-hTERT fusion mRNA and henceprotein.

In summary:

1. The VP22-hTERT, VP22-hTERT-(cMyc-HIS-TAG), and VP22-cMyc-(HIS-TAG)fusion genes were verified by sequencing reactions to be free ofsequence mistakes and to be correctly in-frame fused between the VP22and hTERT coding domains, or VP22, hTERT, cMyc-TAG, and HIS-TAG codingdomains, or VP22, cMyc, and HIS-TAG coding domains, respectively.

2. The VP22-hTERT, VP22-hTERT-(cMyc-HIS-TAG), and VP22-cMyc-(HIS-TAG)fusion genes are all capable of expressing intact fusion proteins asdemonstrated by ICC and Western blot analysis from transientlytransfected COS cells.

3. The telomerase catalytic activities of VP22-hTERT andVP22-hTERT-(cMyc-HIS-TAG) fusion proteins were conclusively demonstratedin vitro by TRAP assays.

4. The expression of VP22-hTERT mRNA by RT-PCR and the immortalizedphenotype by population study analysis, successfully demonstrates thatthe VP22-hTERT fusion protein is functional with respect to primaryfibroblast cell immortalization.

EQUIVALENTS

Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting as to the precise form of thedisclosed invention or to the scope of the appended claims which follow.In particular, various substitutions, alterations, and modificationsknown to persons skilled in the art may be made to the invention withoutdeparting from the spirit and scope of the invention as defined by theclaims.

8 1 40 DNA Artificial Sequence Description of Artificial Sequence PCRPRIMER 1 atatatgcta gcgccaccat gccgcgcgct ccccgctgcc 40 2 41 DNAArtificial Sequence Description of Artificial Sequence PCR PRIMER 2atatatgaat tcagtccagg atggtcttga agtctgaggg c 41 3 5 PRT Herpes simplextype 1 virus 3 Arg Ser Ala Ser Arg 1 5 4 5 PRT Herpes simplex type 1virus 4 Arg Thr Ala Ser Arg 1 5 5 5 PRT Herpes simplex type 1 virus 5Arg Ser Arg Ala Arg 1 5 6 5 PRT Herpes simplex type 1 virus 6 Arg ThrArg Ala Arg 1 5 7 5 PRT Herpes simplex type 1 virus 7 Ala Thr Ala ThrArg 1 5 8 6 PRT Herpes simplex type 1 virus 8 Arg Ser Ala Ala Ser Arg 15

We claim:
 1. A fusion polypeptide, comprising: (i) a first partcomprising a translocation moiety, said translocation moiety comprisinga transport function, said translocation moiety selected from the groupconsisting of: (a) a polypeptide comprising a herpesviral VP22 protein;(b) a polypeptide comprising a human immunodeficiency virus (HIV) TATprotein; (c) a polypeptide comprising a homeodomain of an Antennapediaprotein (Antp HD); (d) a polypeptide comprising an Arginine repeat (Argrepeat); and (e) a cationic polymer; and (ii) a second part comprising apolypeptide selected from the group consisting of: (a) a polypeptidehaving cell immortalization activity, (b) a polypeptide that synthesizestelomeric DNA at chromosomal ends, and (c) a polypeptide which is atranscriptional activator of telomerase activity resulting in synthesisof telomeric DNA at chromosomal ends.
 2. The fusion polypeptide of claim1, wherein the polypeptide having cell immortalization activity isselected from the group consisting of: SV40 small T antigen, SV40 largeT antigen, adenovirus E1A, papilloma virus E6, papilloma virus E7,Epstein-Barr virus, Epstein-Barr nuclear antigen-2, human T-cellleukemia virus-1 (HTLV-1), HTLV-1 tax, herpesvirus saimiri, mutant p53,myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src, c-fgr, myb, c-myc, n-mye,v-myc, and Mdm2.
 3. The fusion polypeptide of claim 1, wherein thepolypeptide that synthesizes telomeric DNA at chromosomal ends isselected from the group consisting of telomerase, telomerase reversetranscriptase (TERT), p140, p105, p48, and p43.
 4. A method oftransiently immortalizing a cell comprising the step of: (a) expandingthe cell in growth medium containing: (i) a first part comprising atranslocation moiety, said translocation moiety comprising a transportfunction, said translocation moiety selected from the group consistingof: (a) a polypeptide comprising a herpesviral VP22 protein; (b) apolypeptide comprising a human immunodeficiency virus (HIV) TAT protein;(c) a polypeptide comprising a homeodomain of an Antennapedia protein(Antp HD); (d) a polypeptide comprising an Arginine repeat (Arg repeat);and (e) a cationic polymer; and (ii) a second part comprising apolypeptide having cell immortalization activity; wherein (b) the fusionprotein is taken up by the cell to result in proliferation of the cell;and (c) wherein growing the immortalized cell in growth medium that doesnot contain the fusion protein terminates the proliferative effects ofthe fusion protein.
 5. The method of claim 4, wherein the secondpolypeptide having cell immortalization activity is selected from thegroup consisting of: SV40 small T antigen, SV40 large T antigen,adenovirus E1A, papilloma virus E6, papilloma virus E7, Epstein-Barrvirus, Epstein-Barr nuclear antigen-2, human T-cell leukemia virus-1(HTLV-1), HTLV-1 tax, herpesvirus saimiri, mutant p53, myc, c-jun,c-ras, c-Ha-ras, h-ras, v-src, c-fgr, myb, c-myc, n-myc, v-myc, andMdm2.
 6. A method of transiently increasing the replicative capacity ofa cell comprising of the steps of: (a) exposing the cell to growthmedium containing a fusion protein comprising: (i) a first partcomprising a translocation moiety, said translocation moiety comprisinga transport function, said translocation moiety selected from the groupconsisting of: (a) a polypeptide comprising a herpesviral VP22 protein;(b) a polypeptide comprising a human immunodeficiency virus (HIV) TATprotein; (c) a polypeptide comprising a homeodomain of an Antennapediaprotein (Antp HD); (d) a polypeptide comprising an Arginine repeat (Argrepeat); and (e) a cationic polymer; and (ii) a second part comprising apolypeptide that synthesizes telomeric DNA at chromosomal ends; then (b)exposing the cell to growth medium that does not contain the fusionprotein of step (a); (c) wherein growing the cell in growth medium thatdoes not contain the fusion protein terminates the replicativecapacity-increasing effects of the fusion protein.
 7. The method ofclaim 6, wherein the second polypeptide is selected from the groupconsisting of: telomerase, telomerase reverse transcriptase (TERT),p140,p105, p48, and p43.
 8. A method of transiently proliferating a celland increasing the replicative capacity of such cell comprising of thesteps of: (I) expanding the cell in growth medium containing: (A) afirst fusion protein comprising: (i) a firs t part comprising atranslocation moiety, said translocation moiety comprising a transportfunction, said translocation moiety selected from the group consistingof: (a) a polypeptide comprising a herpesviral VP22 protein; (b) apolypeptide comprising a human immunodeficiency virus (HIV) TAT protein;(c) a polypeptide comprising a homeodomain of an Antennapedia protein(Antp HD); (d) a polypeptide comprising an Arginine repeat (Arg repeat);and (e) a cationic polymer; and (ii) a second part comprising apolypeptide having cell immortalization activity; and (B) a secondfusion protein comprising: (i) a first part comprising a translocationmoiety, said translocation moiety comprising a transport function, saidtranslocation moiety selected from the group consisting of: (a) apolypeptide comprising a herpesviral VP22 protein; (b) a polypeptidecomprising a human immunodeficiency virus (HIV) TAT protein; (c) apolypeptide comprising a homeodomain of an Antennapedia protein (AntpHD); (d) a polypeptide comprising an Arginine repeat (Arg repeat); and(e) a cationic polymer; and (ii) a second part comprising a polypeptidethat synthesizes telomeric DNA at chromosomal ends; then (II) exposingthe cell to growth medium that does not contain the fusion proteins ofstep (I); wherein (III) growing the cell in growth medium of step (II)terminates the replicative capacity-increasing effects of the fusionproteins.
 9. The method of claim 8, wherein the polypeptide having cellimmortalization activity is selected from the group consisting of: SV40small T antigen, SV40 large T antigen, adenovirus E1A, papilloma virusE6, papilloma virus E7, Epstein-Barr virus, Epstein-Barr nuclearantigen-2, human T-cell leukemia virus-1 (HTLV-1), HTLV-1 tax,herpesvirus saimiri, mutant p53, myc, c-jun, c-ras, c-Ha-ras, h-ras,v-src, c-fgr, myb, c-myc, n-myc, v-myc, and Mdm2.
 10. The method ofclaim 8, wherein the second polypeptide is selected from the groupconsisting of: telomerase, telomerase reverse transcriptase (TERT),p140, p105, p48, and p43.
 11. A cell culture medium containing at leastone fusion protein of claim
 1. 12. A production cell that expresses atleast one fusion protein of claim 1.